3.2 Focus on species
Our results are broadly congruent with the known distribution of the French freshwater bivalve species (Prié, 2017). However, some species were detected out of their known distribution range, noticeably introduced and invasive species, but not only. Others were not detected where they were expected, and valuable data was collected about poorly known species.
3.2.1 Out-of-range species
Outstanding data has been collected with eDNA for conservation-concerned species, such as the Thick-shelled River Mussel U. crassus(categorized EN by the IUCN and listed in the European habitat directive annex II). The Thick-shelled River Mussel is widespread in the basins of the Loire, Saône, Seine, and generally speaking in the north-eastern half of France as shown in Figure 3a. Although its populations have largely declined over the last century (Lopes-Lima, Kebapçı, & Van Damme, 2014), there are many recent records of living populations throughout its range. On the other hand, the species was not known south of the Loire catchment area on the Atlantic coast (except for a few specimens in museum collections from the Dordogne with doubtful vouchers, unpublished data) and historical records downstream of the Rhône catchment area have not been updated despite intensive research in the Camargue wetland and Ardèche River. Data collected by eDNA analysis confirmed the presence of the species throughout the north-eastern quarter of its range and revealed its presence (i) upstream of the Dordogne and Charente watersheds, south of the Loire drainage, where field surveys confirmed its presence by the observation of a few live individuals in 2018 and 2019, and (ii) in several sectors of the Rhône River mainstream, particularly downstream, where it was not known from recent data.
The Rhône River is one of the largest European rivers. The lower Rhône, not surprisingly, remains a terra incognita for the malacologist. It is a large, fast-flowing, deep, turbid, navigated river that is very hard to sample. No scuba-diving nor extensive dredging campaign has been performed to investigate freshwater bivalves species. The lower Rhône flows in the Mediterranean area, and its valley constitutes a northward extension of the northwestern Mediterranean subregion (ex. Darwall et al., 2014). Its tributaries in the lower part are typical Mediterranean streams on its right bank and rather alpine streams on its left bank. Hence its tributaries, which are easier to sample, are very different ecosystems from the Rhône itself. While the Rhône valley allows a northward extension of the Mediterranean biome, the Rhône itself provides a southward path for species of more northern affinities such as the Thick-shelled River Mussel and the Swollen River Mussel U. tumidus Philipsson, 1788. These new data could justify the implementation of conservation measures (Natura 2000 network in particular) on new stretches of the river.
Species of the genus Anodonta are sometimes difficult to identify and are probably often misidentified in databases. Because these species are widespread and common, validation processes of databases do not focus on these potential misidentifications. In many cases, a barcoding approach is needed to clarify the Anodonta populations’ taxonomic status. As an example, the Italian Swan Mussel Anodonta exulcerata Porro, 1838 has been recently re-described in Italy, based on molecular data (Froufe et al., 2017). There is no reliable diagnostic morphological character to identify it in the field (Riccardi et al., 2019). It was considered endemic to Italy and Switzerland. In this study, eDNA sequences of the species were detected in a few places in the upper Loire River. The Italian Swan Mussel is a new species for France, maybe introduced from Italy. It would for sure have been overlooked without an eDNA highlighting.
3.2.2 Invasive species
The Chinese Pond Mussel Sinanodonta woodiana (Lea, 1834) is a large species, introduced in France since 2007, which has rapidly spread in the south of France and the Rhone basin (Adam, 2010). Data collected by eDNA analysis confirmed the expansion of this easily identifiable species in the field, and add a few localities that were not previously known: downstream of the Tarn River, confirming the rapid colonization of the Garonne basin, the Sèvre niortaise River, from where it has since been observed with traditional methods, the Saône River, where it had not been detected during the freshwater bivalves surveys carried out in 2016 by scuba-diving, and the Moselle River. On the other hand, eDNA analyses show that it is probably still absent from the Meuse and Dordogne watersheds, where the eDNA observation pressure is high, and apparently from the Adour, Seine and Charente basins. For all these basins, it would be urgent to implement protection measures to avoid the colonization of the Chinese Pond Mussel. Indeed, it is now known that this species represents a significant threat to native species (Donrovich et al., 2017; Huber & Geist, 2019).
The Quagga Mussel Dreissena rostriformis bugensis (Andrusov, 1897) is a smaller species, similar in shell shape to the widespread Zebra mussel Dreissena polymorpha (Pallas, 1771), and therefore likely to remain unnoticed due to confusion or negligence. It has only been known in France since 2010 when it was first detected in the Meuse and Moselle basins (Bij de Vaate & Beisel, 2011). According to previously available data shown in Figure 3b, it was thought to have colonized only the Meuse and Moselle Rivers, with scattered data in the Rhine and Rhône Rivers (Prié & Fruget, 2017). eDNA data shows that the species has already spread considerably, being also introduced into the Mediterranean coastal rivers (Lez River in Montpellier, probably via the Bas-Rhône-Languedoc Canal), and into the Seine watershed (probably via the Canal de Bourgogne), where it had never been observed with traditional surveys. None of our samples from the Atlantic drainages contained Quagga Mussel eDNA. These results highlights the value of eDNA sampling for invasive species’ early detection and monitoring.
The Ridgedbeak Peaclam Euglesa compressa (Prime, 1852) has been formally discovered in France very recently (Mouthon & Forcellini, 2017), although the first data on its distribution date from 1989 (Mouthon & Taïr Abacci, 2012). Analysis of the eDNA has extended its current range, with new data in the Meuse, upstream of the Seine, the Saône, the Ardèche, and all along the lower Rhône River.
3.2.3 Rare and endangered species
Our large-scale sampling in France stressed out the worrying conservation status of some species which are not currently considered of conservation concern. The Depressed River Mussel P. complanatahistorically had a wide distribution in France, as evidenced by museum collections (especially those of the National Museum of Natural History in Paris, unpublished data), where three subspecies were recognized (Bouchet, 2002). About a hundred records are available in France (see Figure 3c). Out of these, 46 concern individuals observed living recently. These data attest to the presence of the species in the catchment areas of the Seine (about 15 stations), the Somme (one station), the Meuse (two stations), the Moselle (one station) and the Rhine (one station). There is no recent record of live specimens elsewhere in France. eDNA data confirm the current presence of the species in the Seine, the Meuse (9 stations out of the 52 sampled), the Moselle (only one station studied), but also in the Saône (one station out of the two studied), the lower Rhône (only one station out of the 65 sampled) and the Charente (only one station out of the 7 analyzed) Rivers. Depressed River Mussel eDNA was not detected in any of the localities sampled in potentially favorable sites (downstream ecosystems on limestone substratum) in the Garonne basin. Similarly, although few favorable stations were studied on the Loire drainage, none revealed the species. The Depressed River Mussel seems to be extirpated from most of its range in France. The only data on the lower Rhône is remarkable, as no historical data are known for this sector.
The Witham Orb Mussel Sphaerium solidum is a typical downstream species, living in large rivers, at depth, in well-oxygenated water, preferably on sandy substrates. This kind of environment has been extensively sampled in our study (estimated 250 sampling sites suitable to the Witham Orb Mussel), but the Witham Orb Mussel has been detected only once in our samples, close to one of the three places where it has been last observed alive. Comprehensive data is available given its easily identifiable thick shell, which can be preserved for years in the river banks as shown in Figure 3d. But this data, mainly based on shell observations, probably give an obsolete picture of the reality. According to eDNA results, the Witham Orb Mussel appears to be extirpated from the Aisne site by now (where sampling occurred about 10 years after its observation). Only a single population would remain, in the lower Seine, which makes the Witham Orb Mussel the most threatened freshwater bivalve species in France.
The Nut Orb Mussel S. rivicola is also a species characteristic of downstream large rivers, probably less demanding in terms of the substrate than the Witham Orb Mussel, preferring more muddy sediments. It also seems a little less threatened, but its situation concerning eDNA sampling throughout France is alarming. About a hundred records are available, spread over most of the large French catchment areas. Nevertheless, very few living populations are currently known. At least one population seems to subsist downstream of the Loire, on a sector that we did not sample in the framework of this work. eDNA was only detected in a single site in the Rhône catchment (out of 65 sampled sites) eDNA could be detected indicating that Nut Orb Mussels still occurs there, but probably with very poor conservation status since it is not known elsewhere in the basin. Living individuals have recently been collected on the Meuse River, data confirmed by eDNA analysis. Finally, there are several recent records of living individuals in the Seine basin, but the eDNA sampling revealed the species only once in the Seine basin, closed to a previously reported living population.
The False Orb Pea Mussel Euglesa pseudosphaerium Favre, 1927 is a lowland rivers species that lives on stable swampy areas, in clear and clean waters and is intolerant to pollution and water level fluctuations (Welter-Schultes 2012). This kind of habitat has become very rare in France, and so is the False Orb Pea Mussel. Welter-Schultes (2012) gives it a wide distribution range, covering most of France, although it is rare and localized everywhere. Mouthon and Kuiper (1987) recorded it from only four sites in the Rhone Valley and report an additional historical record near Toulouse (south-west France). Despite extensive sampling, we didn’t detect its eDNA in the Rhone Valley. It has been detected with eDNA analyses once in a small tributary of the Charente River and once in Britany (north-west France). Although suitable habitat for this species is under-sampled, eDNA data confirm the worrying conservation status of the False Orb Pea Mussel.